RESUMEN
κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion.
Asunto(s)
Unión Proteica , Proteínas de Unión al GTP ral , Proteínas ras , Humanos , Proteínas de Unión al GTP ral/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Mutación , Guanosina Trifosfato/metabolismo , Transducción de Señal , HidrólisisRESUMEN
BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.
Asunto(s)
Sistemas CRISPR-Cas , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Ratones , Animales , GTP Fosfohidrolasas/metabolismo , Edición Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Carcinogénesis/genética , Células Madre Neoplásicas/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
RALA and RALB are highly homologous small G proteins belonging to the RAS superfamily. Like other small GTPases, the RALs are molecular switches that can be toggled between inactive GDP-bound and active GTP-bound states to regulate diverse and critical cellular functions such as vesicle trafficking, filopodia formation, mitochondrial fission, and cytokinesis. The RAL paralogs are activated and inactivated by a shared set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) and utilize similar sets of downstream effectors. In addition to their important roles in normal cell biology, the RALs are known to be critical mediators of cancer cell survival, invasion, migration, and metastasis. However, despite their substantial similarities, the RALs often display striking functional disparities in cancer. RALA and RALB can have redundant, unique, or even antagonistic functions depending on cancer type. The molecular basis for these discrepancies remains an important unanswered question in the field of cancer biology. In this review we examine the functions of the RAL paralogs in normal cellular physiology and cancer biology with special consideration provided to situations where the roles of RALA and RALB are non-redundant.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Neoplasias , Supervivencia Celular , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
BACKGROUND AND AIMS: Ras-like (Ral) small guanosine triphosphatases (GTPases), RalA and RalB, are proto-oncogenes directly downstream of Ras and cycle between the active guanosine triphosphate-bound and inactive guanosine diphosphate-bound forms. RalGTPase-activating protein (RalGAP) complex exerts a negative regulation. Currently, the role of Ral up-regulation in cancers remains unclear. We aimed to examine the clinical significance, functional implications, and underlying mechanisms of RalA signaling in HCC. APPROACH AND RESULTS: Our in-house and The Cancer Genome Atlas RNA sequencing data and quantitative PCR data revealed significant up-regulation of RalA in patients' HCCs. Up-regulation of RalA was associated with more aggressive tumor behavior and poorer prognosis. Consistently, knockdown of RalA in HCC cells attenuated cell proliferation and migration in vitro and tumorigenicity and metastasis in vivo. We found that RalA up-regulation was driven by copy number gain and uncovered that SP1 and ETS proto-oncogene 2 transcription factor cotranscriptionally drove RalA expression. On the other hand, RalGAPA2 knockdown increased the RalA activity and promoted intrahepatic and extrahepatic metastasis in vivo. Consistently, we observed significant RalGAPA2 down-regulation in patients' HCCs. Intriguingly, HCC tumors showing simultaneous down-regulation of RalGAPA2 and up-regulation of RalA displayed a significant association with more aggressive tumor behavior in terms of more frequent venous invasion, more advanced tumor stage, and poorer overall survival. Of note, Ral inhibition by a Ral-specific inhibitor RBC8 suppressed the oncogenic functions in a dose-dependent manner and sensitized HCC cells to sorafenib treatment, with an underlying enhanced inhibition of mammalian target of rapamycin signaling. CONCLUSIONS: Our results provide biological insight that dysregulation of RalA signaling through dual regulatory mechanisms supports its oncogenic functions in HCC. Targeting RalA may serve as a potential alternative therapeutic approach alone or in combination with currently available therapy.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas de Unión al GTP ral , Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Regulación hacia Abajo , Proteínas Activadoras de GTPasa/genética , Humanos , Neoplasias Hepáticas/genética , Transducción de Señal , Proteínas de Unión al GTP ral/genéticaRESUMEN
RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.
Asunto(s)
Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Receptores ErbB/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Receptores de Péptidos de Invertebrados/metabolismo , Células Madre/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Animales , Animales Modificados Genéticamente , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endocitosis , Receptores ErbB/genética , Femenino , Humanos , Hiperplasia , Mucosa Intestinal/patología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glándulas Mamarias Humanas/enzimología , Glándulas Mamarias Humanas/patología , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Receptores de Péptidos de Invertebrados/genética , Transducción de Señal , Células Madre/patología , Proteínas de Unión al GTP ral/genéticaRESUMEN
BACKGROUND: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. METHODS: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. RESULTS: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. CONCLUSIONS: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.
Asunto(s)
Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteínas de Unión al GTP ral/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , Paclitaxel/uso terapéutico , Pronóstico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/genéticaRESUMEN
Characterizing the consequences of mutated Ras/LET-60 on the development of the C. elegans vulva has provided critical insights into the role of Ras in normal animal development. Furthermore, double mutant analysis revealed the role of Ras relative to other components of growth factor signal transduction. Here we describe the combined use of principles of parallelism and epistasis to investigate the use of different Ras effectors, Raf and RalGEF > Ral, during the development of the vulva and other tissues. We additionally describe the use of these principles to delineate the function of the close Ras relative, RAP-1. The worm continues to lead the way in clarifying otherwise poorly understood functions of Ras during animal development.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Vulva/crecimiento & desarrollo , Proteínas de Unión al GTP ral/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Femenino , Transducción de Señal , Vulva/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP rap1/genética , Proteínas ras/genéticaRESUMEN
Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.
Asunto(s)
Adenocarcinoma/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Regiones no Traducidas 5' , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/tratamiento farmacológico , Animales , Línea Celular Tumoral , Cicloheximida/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , G-Cuádruplex , Genes ras/genética , Humanos , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Oxidación-Reducción , Neoplasias Pancreáticas/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Helicasas , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Triterpenos/farmacología , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, cross talk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent nuclear factor of activated T cells nuclear translocation, suggesting that it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Fototransducción/genética , Optogenética/métodos , Proteína de Unión al GTP rhoA/genética , Animales , Técnicas Biosensibles/métodos , Diferenciación Celular , Proliferación Celular , Perros , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Luz , Células de Riñón Canino Madin Darby , Especificidad de Órganos , Fosfoinositido Fosfolipasa C/genética , Fosfoinositido Fosfolipasa C/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Micro-ribonucleic acids (miRNAs) are a class of conserved small non-coding RNAs (sncRNAs) that post-transcriptionally regulate their downstream target genes. Existing evidence indicates that abnormal expression of mRNAs results in the occurrence and development of pancreatic cancer (PC). In this study, we explored the potential role of miRNA-139 (miR-139) as a biomarker in the monitoring and treatment of PC. We demonstrated that expression of miR-139 was significantly downregulated in PC cells and tissues. In addition, both in vitro and in vivo experiments showed that miR-139 significantly inhibited the growth, migration, and invasion of PC cells. We carried out microarray analysis and transcriptome sequencing to find the potential target of miR-139 in PC cells, and the results showed that miR-139 targeted Ras-like proto-oncogene B (RalB). Luciferase reporter experiments verified that high level of RalB could reverse the proliferation and invasion of PC cells overexpressing miR-139. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we found that miR-139 likely affected PC cell cycle by targeting RalB via the Ral/protein kinase B (Akt) serine/threonine kinase 1 (RAC)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway, thus affecting cell proliferation. This presumption was further confirmed in our in vitro and in vivo experiments. Our examination of PC tissues suggested that the expression of miR-139 was negatively correlated with that of RalB. Taken together, our results implied that miR-139 could suppress tumor growth and metastasis in PC by targeting RalB, revealing the potential role of miR-139 as a biomarker for the monitoring and treatment of PC.
Asunto(s)
Carcinogénesis/metabolismo , MicroARNs/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Neoplásico/metabolismo , Transducción de Señal , Proteínas de Unión al GTP ral/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Células HEK293 , Humanos , MicroARNs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/genética , ARN Neoplásico/genética , Proteínas de Unión al GTP ral/genéticaRESUMEN
Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.
Asunto(s)
GTP Fosfohidrolasas , Mieloma Múltiple , Supervivencia Celular/genética , Genes ras , Humanos , Mieloma Múltiple/genética , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
RAS mutant (MT) metastatic colorectal cancer (mCRC) is resistant to MEK1/2 inhibition and remains a difficult-to-treat group. Therefore, there is an unmet need for novel treatment options for RASMT mCRC. RALA and RALB GTPases function downstream of RAS and have been found to be key regulators of several cell functions implicated in KRAS-driven tumorigenesis. However, their role as regulators of the apoptotic machinery remains to be elucidated. Here, we found that inhibition of RALB expression, but not RALA, resulted in Caspase-8-dependent cell death in KRASMT CRC cells, which was not further increased following MEK1/2 inhibition. Proteomic analysis and mechanistic studies revealed that RALB depletion induced a marked upregulation of the pro-apoptotic cell surface TRAIL Death Receptor 5 (DR5) (also known as TRAIL-R2), primarily through modulating DR5 protein lysosomal degradation. Moreover, DR5 knockdown or knockout attenuated siRALB-induced apoptosis, confirming the role of the extrinsic apoptotic pathway as a regulator of siRALB-induced cell death. Importantly, TRAIL treatment resulted in the association of RALB with the death-inducing signalling complex (DISC) and targeting RALB using pharmacologic inhibition or RNAi approaches triggered a potent increase in TRAIL-induced cell death in KRASMT CRC cells. Significantly, high RALB mRNA levels were found in the poor prognostic Colorectal Cancer Intrinsic Subtypes (CRIS)-B CRC subgroup. Collectively, this study provides to our knowledge the first evidence for a role for RALB in apoptotic priming and suggests that RALB inhibition may be a promising strategy to improve response to TRAIL treatment in poor prognostic RASMT CRIS-B CRC.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteínas de Unión al GTP ral/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bencimidazoles/administración & dosificación , Neoplasias Colorrectales/genética , Humanos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Transfección , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/biosíntesis , Proteínas de Unión al GTP ral/genéticaRESUMEN
The C. elegans vulva is an excellent model for the study of developmental biology and cell-cell signaling. The developmental induction of vulval precursor cells (VPCs) to assume the 3°-3°-2°-1°-2°-3° patterning of cell fates occurs with 99.8% accuracy. During C. elegans vulval development, an EGF signal from the anchor cell initiates the activation of RasLET-60 > RafLIN-45 > MEKMEK-2 > ERKMPK-1 signaling cascade to induce the 1° cell. The presumptive 1° cell signals its two neighboring cells via NotchLIN-12 to develop 2° cells. In addition, RasLET-60 switches effectors to RalGEFRGL-1 > RalRAL-1 to promote 2° fate. Shin et al. (2019) showed that RalGEFRGL-1 is a dual-function protein in VPCs fate patterning. RalGEFRGL-1 functions as a scaffold for PDKPDK-1 > AktAKT-1/2 modulatory signaling to promote 1° fate in addition to propagating the RasLET-60 modulatory signal through RalRAL-1 to promote 2° fate. The deletion of RalGEFRGL-1 increases the frequency of VPC patterning errors 15-fold compared to the wild-type control. We speculate that RalGEFRGL-1 represents an "insulated switch", whereby the promotion of one signaling activity curtails the promotion of the opposing activity. This property might increase the impact of the switch on fidelity more than two separately encoded proteins could. Understanding how developmental fidelity is controlled will help us to better understand the origins of cancer and birth defects, which occur in part due to the misspecification of cell fates.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Transducción de Señal , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Linaje de la Célula , Femenino , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vulva/citología , Vulva/crecimiento & desarrollo , Vulva/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The NLRP3 inflammasome is an important mediator of inflammatory responses and its regulation is an active area of research. RalA is a Ras-like GTPase, which play pivotal roles in the biology of cells. So far, there have been very few studies on RalA regulating inflammatory responses. Bioinformatics analysis predicted that RalA might participate in the regulatory network of NLRP3 inflammasome, which has been confirmed in THP-1 macrophages. After virtual screening of compounds, it was found that levonidazole selected from our virtual small molecule compound library has the potential to bind to RalA. Of note, the interaction of RalA/levornidazole was verified by Surface Plasmon Resonance-Biacore T200, LC/MS analysis and Western blotting analysis. Molecular dynamics simulations revealed that the conformational changes of RalA might be regulated by levornidazole. Additionally, IL-1ß/IL-18 secretion from ATP + LPS stimulated THP-1-derived macrophages was RalA-dependently suppressed by levornidazole, suggesting that RalA might have an inhibitory effect on NLRP3 inflammasome activation. The results of co-immunoprecipitation and RalA depletion experiments showed that levornidazole could induce RalA to block the assembly of NLRP3/ASC/pro-caspase-1 complex, thereby reducing the levels of cleaved-caspase-1 and IL-1ß/IL-18 secretion. Our study has suggested an anti-inflammatory function of RalA and identified its targeting chemical compound. Overall, this study clarifies a novel pharmacological mechanism by which RalA/levornidazole inhibits NLRP3 inflammasome activation and IL-1ß/IL-18 secretion.
Asunto(s)
Inflamasomas/inmunología , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Macrófagos/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Ornidazol/farmacología , Proteínas de Unión al GTP ral/genética , Animales , Femenino , Humanos , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Células THP-1RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.
Asunto(s)
Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , GTP Fosfohidrolasas/genética , Neoplasias Pancreáticas/genética , Pancreatitis/genética , Proteínas/genética , Células Acinares/patología , Anciano , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Femenino , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Estimación de Kaplan-Meier , Masculino , Metaplasia/genética , Metaplasia/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pancreatitis/metabolismo , Proteínas/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter-reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)-regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21Waf1/cip1 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase-related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21Waf1/cip1 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.
Asunto(s)
Antioxidantes/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Humanos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.
Asunto(s)
Adipocitos/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Ratones , Microscopía Fluorescente , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
FK506-binding protein-like (FKBPL) has previously been shown to inhibit angiogenesis viain vitro and in vivo experimentation. Thus, it was proposed that the delivery of a siRNA targeting FKBPL could hold great potential in promoting angiogenesis for advanced wound healing applications. An effective delivery system has been utilised to encapsulate the siFKBPL to form nanoparticles, thereby improving cellular entry and eliciting a potent angiogenic response. In this study, nanoparticles were formed via condensation of siFKBPL with RALA; a novel, cationic 30 mer amphipathic peptide. Nanoparticles prepared at a N:P ratio of 6 demonstrated an average particle size of 76.6nm with a zeta potential of +16.5mV. Treatment of HMEC-1 cells at N:P 6 resulted in a transfection efficiency of 33.7%, negligible cytotoxicity, and significant knockdown of endogenous FKBPL expression. Functionally, treatment with RALA/siFKBPL resulted in significant improvements in cell migration and endothelial tubule formation in vitro. The process of electrospinning was employed to fabricate a nanofibrous wound patch to facilitate the controlled delivery of the RALA/siFKBPL nanoparticles. Alginate/poly-(vinyl alcohol) was electrospun following electrospinning of Chitosan/poly-(vinyl alcohol) to form a bilayered wound patch. Subsequently, the nanofibres were crosslinked to improve stability, before nanoparticle incorporation via soak loading. In vivo wound healing studies using C57BL/6J mice demonstrated a significant increase in angiogenesis when the RALA/siFKBPL nanoparticles were delivered from the bilayered wound patch; a 326% increase in blood vessel density was observed compared to untreated wounds. Taken together, this data demonstrates that delivery of RALA/siFKBPL nanoparticles from the bilayered wound patch represents an innovative wound healing therapy.
Asunto(s)
Nanopartículas , Proteínas de Unión a Tacrolimus/genética , Cicatrización de Heridas/genética , Proteínas de Unión al GTP ral/genética , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Ratones , Ratones Endogámicos C57BL , Nanofibras , Neovascularización Fisiológica/genética , Tamaño de la Partícula , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
The field of gene therapy still attracts great interest due to its potential therapeutic effect towards the most deadly diseases, such as cancer. For cancer gene therapy to be feasible and viable in a clinical setting, the design and development of a suitable gene delivery system is imperative. Peptide based vectors, in particular, reveal to be promising for therapeutic gene release. Following this, two different peptides, RALA and WRAP5, have been investigated mainly regarding their ability to form complexes with a p53 encoding plasmid (pDNA) with suitable properties for gene delivery. To address this issue, and after an initial screening study focused on the dependence of pDNA complexation capacity with the nitrogen to phosphate groups (N/P) ratio, a design of experiments (DoE) tool has been employed. For each peptide/pDNA system, parameters such as, the buffer pH and the N/P ratio were considered the DoE inputs and the vector size, zeta potential and pDNA complexation capacity (CC) were monitored as DoE outputs. The main goal was to find the optimal experimental conditions to minimize particle sizes, as well as, to maximize the positive surface charges of the formulated nanosystems and maximize the pDNA CC. Through the DoE method applied, the optimal RALA/pDNA and WRAP5/pDNA formulations were revealed and show interesting features related to peptide structure and pDNA complexation ability. This work illustrates the great utility of experimental design tools in optimizing the formulation of peptide/pDNA vectors in a minimum number of experiments providing relevant knowledge for the development of more suitable and efficient gene delivery systems. The new insights achieved on these carriers clearly instigate deeper research on gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Péptidos/genética , Plásmidos/química , Proteína p53 Supresora de Tumor/genética , Proteínas de Unión al GTP ral/genética , Secuencia de Aminoácidos , Análisis Factorial , Terapia Genética/métodos , Humanos , Concentración de Iones de Hidrógeno , Neoplasias/genética , Neoplasias/terapia , Péptidos/síntesis química , Péptidos/metabolismo , Plásmidos/metabolismo , Unión Proteica , Electricidad Estática , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Protein lysine fatty acylation is increasingly recognized as a prevalent and important protein post-translation modification. Recently, it has been shown that K-Ras4a, R-Ras2, and Rac1 are regulated by lysine fatty acylation. Here, we investigated whether other members of the Ras superfamily could also be regulated by lysine fatty acylation. Several small GTPases exhibit hydroxylamine resistant fatty acylation, suggesting they may also have protein lysine fatty acylation. We further characterized one of these GTPases, RalB. We show that RalB has C-terminal lysine fatty acylation, with the predominant modification site being Lys200. The lysine acylation of RalB is regulated by SIRT2, a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacylases. Lysine fatty acylated RalB exhibited enhanced plasma membrane localization and recruited its known effectors Sec5 and Exo84, members of the exocyst complex, to the plasma membrane. RalB lysine fatty acylation did not affect the proliferation or anchorage-independent growth but did affect the trans-well migration of A549 lung cancer cells. This study thus identified an additional function for protein lysine fatty acylation and the deacylase SIRT2.